4-(8-methoxy-1-((1-methoxypropan-2-yl)-2-(tetrahydro-2h-pyran-4-yl)-1 h-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole and its use as bromodomain inhibitor

ABSTRACT

Novel quinoline compounds pharmaceutical compositions containing such compounds and their use in therapy.

FIELD OF THE INVENTION

The present invention relates to novel compounds, pharmaceuticalcompositions containing such compounds and to their use in therapy.

BACKGROUND OF THE INVENTION

The genomes of eukaryotic organisms are highly organised within thenucleus of the cell. The long strands of duplex DNA are wrapped aroundan octomer of histone proteins (most usually comprising two copies ofhistones H2A, H2B H3 and H4) to form a nucleosome. This basic unit isthen further compressed by the aggregation and folding of nucleosomes toform a highly condensed chromatin structure. A range of different statesof condensation are possible, and the tightness of this structure variesduring the cell cycle, being most compact during the process of celldivision. Chromatin structure plays a critical role in regulating genetranscription, which cannot occur efficiently from highly condensedchromatin. The chromatin structure is controlled by a series of posttranslational modifications to histone proteins, notably histones H3 andH4, and most commonly within the histone tails which extend beyond thecore nucleosome structure. These modifications include acetylation,methylation, phosphorylation, ubiquitinylation, SUMOylation. Theseepigenetic marks are written and erased by specific enzymes, which placethe tags on specific residues within the histone tail, thereby formingan epigenetic code, which is then interpreted by the cell to allow genespecific regulation of chromatin structure and thereby transcription.

Histone acetylation is most usually associated with the activation ofgene transcription, as the modification loosens the interaction of theDNA and the histone octomer by changing the electrostatics. In additionto this physical change, specific proteins bind to acetylated lysineresidues within histones to read the epigenetic code. Bromodomains aresmall (˜110 amino acid) distinct domains within proteins that bind toacetylated lysine resides commonly but not exclusively in the context ofhistones. There is a family of around 50 proteins known to containbromodomains, and they have a range of functions within the cell.

The BET family of bromodomain containing proteins comprises 4 proteins(BRD2, BRD3, BRD4 and BRD-t) which contain tandem bromodomains capableof binding to two acetylated lysine residues in close proximity,increasing the specificity of the interaction. BRD2 and BRD3 arereported to associate with histones along actively transcribed genes andmay be involved in facilitating transcriptional elongation (Leroy et al,Mol. Cell. 2008 30(1):51-60), while BRD4 appears to be involved in therecruitment of the pTEF-β complex to inducible genes, resulting inphosphorylation of RNA polymerase and increased transcriptional output(Hargreaves et al, Cell, 2009 138(1): 129-145). It has also beenreported that BRD4 or BRD3 may fuse with NUT (nuclear protein in testis)forming novel fusion oncogenes, BRD4-NUT or BRD3-NUT, in a highlymalignant form of epithelial neoplasia (French et al. Cancer Research,2003, 63, 304-307 and French et al. Journal of Clinical Oncology, 2004,22 (20), 4135-4139). Data suggests that BRD-NUT fusion proteinscontribute to carcinogenesis (Oncogene, 2008, 27, 2237-2242). BRD-t isuniquely expressed in the testes and ovary. All family members have beenreported to have some function in controlling or executing aspects ofthe cell cycle, and have been shown to remain in complex withchromosomes during cell division—suggesting a role in the maintenance ofepigenetic memory. In addition some viruses make use of these proteinsto tether their genomes to the host cell chromatin, as part of theprocess of viral replication (You et al Cell, 2004 117(3):349-60).

Japanese patent application JP2008-156311 discloses a benzimidazolederivative which is said to be a BRD2 bromodomain binding agent whichhas utility with respect to virus infection/proliferation.

Patent application WO2009084693A1 discloses a series ofthienotriazolodiazepiene derivatives that are said to inhibit thebinding between an acetylated histone and a bromodomain containingprotein which are said to be useful as anti-cancer agents.

Patent application WO2011/054846 discloses a series of quinolinederivatives that inhibit the binding of BET family bromodomains withacetylated lysine residues.

Novel compounds have been found which inhibit the binding ofbromodomains with its cognate acetylated proteins, more particularly aclass of compounds that inhibit the binding of BET family bromodomainsto acetylated lysine residues. Such compounds will hereafter be referredto as “bromodomain inhibitors”.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, there is provided a compoundof formula (I) or a salt thereof, more particularly a compound offormula (I) or a pharmaceutically acceptable salt thereof

In a second aspect of the present invention, there is provided apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof and one or morepharmaceutically acceptable carriers, diluents or excipients.

In a third aspect of the present invention, there is provided a compoundof formula (I), or a pharmaceutically acceptable salt thereof for use intherapy, in particular in the treatment of diseases or conditions forwhich a bromodomain inhibitor is indicated.

In a fourth aspect of the present invention, there is provided a methodof treating diseases or conditions for which a bromodomain inhibitor isindicated in a subject in need thereof which comprises administering atherapeutically effective amount of a compound of formula (I) or apharmaceutically acceptable salt thereof.

In a fifth aspect of the present invention, there is provided the use ofa compound of formula (I), or a pharmaceutically acceptable salt thereofin the manufacture of a medicament for the treatment of diseases orconditions for which a bromodomain inhibitor is indicated.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Shows an XRPD pattern of a crystalline form of4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(anhydrous form 1).

FIG. 2: Shows an XRPD pattern of a crystalline form of4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(anhydrous form 2).

FIG. 3: Shows an XRPD pattern of a crystalline form of4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(anhydrous form 3).

FIG. 4: Shows an XRPD pattern of a crystalline form of4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]_(q)uinolin-7-yl)-3,5-dimethylisoxazole (hydrate).

FIG. 5: Shows an XRPD pattern of a crystalline form of4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazolehydrochloride (hydrochloride).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a compound of formula (I) which is4-(8-methoxy-1-(1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole

or a salt thereof.

The compound of formula (I) contains a chiral atom such that opticalisomers, e.g. enantiomers may be formed. Accordingly, the presentinvention encompasses all isomers of the compound of formula (I) whetheras individual isomers isolated such as to be substantially free of theother isomer (i.e. pure) or as mixtures (i.e. racemates and racemicmixtures). An individual isomer isolated such as to be substantiallyfree of the other isomer (i.e. pure) may be isolated such that less than10%, particularly less than about 1%, for example less than about 0.1%of the other isomer is present.

Separation of isomers may be achieved by conventional techniques knownto those skilled in the art, e.g. by fractional crystallisation,chromatography or HPLC.

In one embodiment there is provided a compound of formula (IA) which is4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole

or a salt thereof.

In a further embodiment there is provided a compound of formula (IB)which is4-(8-methoxy-1-((S)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole

or a salt thereof.

It will be appreciated that the compounds of formula (IA) and formula(IB) are within the scope of the compound of formula (I). As usedherein, unless otherwise stated, a reference to a compound of formula(I) also includes a reference a the compound of formula (IA) and acompound of formula (IB).

It will be appreciated that the present invention covers compounds offormula (I) as the free base and as salts thereof, for example as apharmaceutically acceptable salt thereof. In one embodiment theinvention relates to compounds of formula (I) in the form of a freebase. In one embodiment the invention relates to compounds of formula(I) or a pharmaceutically acceptable salt thereof.

Because of their potential use in medicine, salts of the compounds offormula (I) are desirably pharmaceutically acceptable. Suitablepharmaceutically acceptable salts can include acid addition salts. For areview of suitable pharmaceutically acceptable salts see Berge et al.,J. Pharm. Sci., 66:1-19, (1977). Typically, a pharmaceuticallyacceptable salt may be readily prepared by using a desired acid or baseas appropriate. The resultant salt may precipitate from solution and becollected by filtration or may be recovered by evaporation of thesolvent.

A pharmaceutically acceptable acid addition salt can be formed byreaction of a compound of formula (I) with a suitable inorganic ororganic acid (such as hydrobromic, hydrochloric, sulphuric, nitric,phosphoric, succinic, maleic, acetic, propionic, fumaric, citric,tartaric, lactic, benzoic, salicylic, glutamaic, aspartic,p-toluenesulfonic, benzenesulfonic, methanesulfonic, ethanesulfonic,naphthalenesulfonic such as 2-naphthalenesulfonic, or hexanoic acid),optionally in a suitable solvent such as an organic solvent, to give thesalt which is usually isolated for example by crystallisation andfiltration or by evaporation followed by trituration. A pharmaceuticallyacceptable acid addition salt of a compound of formula (I) can compriseor be for example a hydrobromide, hydrochloride, sulfate, nitrate,phosphate, succinate, maleate, acetate, propionate, fumarate, citrate,tartrate, lactate, benzoate, salicylate, glutamate, aspartate,p-toluenesulfonate, benzenesulfonate, methanesulfonate, ethanesulfonate,naphthalenesulfonate (e.g. 2-naphthalenesulfonate) or hexanoate salt. Inone embodiment a pharmaceutically acceptable acid addition salt of acompound of formula (I) can comprise or be a hydrochloride, sulfate,maleate, fumarate, citrate, p-toluenesulfonate, benzenesulfonate ormethanesulfonate salt. In a particular embodiment there is provided acompound of formula (IA) in the form of a hydrochloride salt.

Other non-pharmaceutically acceptable salts, e.g. formates, oxalates ortrifluoroacetates, may be used, for example in the isolation of thecompounds of formula (I), and are included within the scope of thisinvention.

The invention includes within its scope all possible stoichiometric andnon-stoichiometric forms of the salts of the compounds of formula (I).

It will be appreciated that many organic compounds can form complexeswith solvents in which they are reacted or from which they areprecipitated or crystallized. These complexes are known as “solvates”.For example, a complex with water is known as a “hydrate”. Solvents withhigh boiling points and/or capable of forming hydrogen bonds such aswater, xylene, N-methylpyrrolidinone, methanol and ethanol may be usedto form solvates. Methods for identification of solvates include, butare not limited to, NMR and microanalysis. Solvates of the compounds offormula (I) are within the scope of the invention.

The invention includes within its scope all possible stoichiometric andnon-stoichiometric forms of the solvates of the compounds of formula(I).

The invention encompasses all prodrugs, of the compound of formula (I)or a pharmaceutically acceptable salt thereof, which upon administrationto the recipient is capable of providing (directly or indirectly) thecompound of formula (I) or a pharmaceutically acceptable salt thereof,or an active metabolite or residue thereof. Such derivatives arerecognizable to those skilled in the art, without undue experimentation.Nevertheless, reference is made to the teaching of Burger's MedicinalChemistry and Drug Discovery, 5^(th) Edition, Vol 1: Principles andPractice, which is incorporated herein by reference to the extent ofteaching such derivatives.

The compounds of formula (I) may be in crystalline or amorphous form.Furthermore, some of the crystalline forms of the compounds of formula(I) may exist as polymorphs, which are included within the scope of thepresent invention. Polymorphic forms of compounds of formula (I) may becharacterized and differentiated using a number of conventionalanalytical techniques, including, but not limited to, X-ray powderdiffraction (XRPD) patterns, infrared (IR) spectra, Raman spectra,differential scanning calorimetry (DSC), thermogravimetric analysis(TGA) and solid state nuclear magnetic resonance (SSNMR).

The compound of formula (IA), that is to say4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole,in the form of a free base has been found to exist in a number ofdifferent crystalline forms, namely, anhydrous forms 1, 2 and 3 andhydrated form 1. Such crystalline forms can be prepared by proceduresdescribed herein.

Thus in one embodiment, there is provided a crystalline form of4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(anhydrous form 1) characterised by substantially the X-ray powderdiffraction (XRPD) pattern as shown in FIG. 1, wherein the XRPD patternis expressed in terms of 2 theta angles and obtained with adiffractometer using copper Kα-radiation using procedures describedherein. The XRPD pattern of anhydrous form 1 shows characteristic 2theta angle peaks as listed in Example 9 Table 1.

In a further embodiment, there is provided a crystalline form of4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(anhydrous form 2) characterised by substantially the X-ray powderdiffraction (XRPD) pattern as shown in FIG. 2, wherein the XRPD patternis expressed in terms of 2 theta angles and obtained with adiffractometer using copper Kα-radiation using procedures described. TheXRPD pattern of anhydrous form 2 shows characteristic 2 theta anglepeaks as listed in Example 9 Table 1.

In a further embodiment, there is provided a crystalline form4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(anhydrous form 3) characterised by substantially the X-ray powderdiffraction (XRPD) pattern as shown in FIG. 3, wherein the XRPD patternis expressed in terms of 2 theta angles and obtained with adiffractometer using copper Kα-radiation using procedures describedherein. The XRPD pattern of anhydrous form 3 shows characteristic 2theta angle peaks as listed in Example 9 Table 1.

In a further embodiment, there is provided a crystalline form of4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(hydrate) characterised by substantially the X-ray powder diffraction(XRPD) pattern as shown in FIG. 4, wherein the XRPD pattern is expressedin terms of 2 theta angles and obtained with a diffractometer usingcopper Kα-radiation using procedures described herein. The XRPD patternof the hydrate shows characteristic 2 theta angle peaks as listed inExample 9 Table 1.

The invention also provides for the compound of formula (IA) that is tosay4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazolehydrochloride in crystalline form. Such a crystalline form can beprepared by procedures described herein.

In a further embodiment, there is provided a crystalline form of4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazolehydrochloride (hydrochloride form 1) characterised by substantially theX-ray powder diffraction (XRPD) pattern as shown in FIG. 5, wherein theXRPD pattern is expressed in terms of 2 theta angles and obtained with adiffractometer using copper Kα-radiation using procedures describedherein. The XRPD pattern of this form shows characteristic 2 theta anglepeaks as listed in Example 9 Table 1.

It will be appreciated from the foregoing that included within the scopeof the invention are solvates, isomers and polymorphic forms of thecompounds of formula (I) and salts thereof.

The compounds of formula (I) or salts thereof may be made by a varietyof methods, including standard chemistry. Illustrative general syntheticmethods are set out below and then specific compounds of formula (I) andpharmaceutically acceptable salts thereof, are prepared in the Examples.

The compound of formula (I) can be prepared from the compound of formula(II) by, for example, heating the compound of formula (II) in aceticacid or p-toluenesulfonic acid in toluene

The compound of formula (II) can be prepared by reaction of the compoundof formula (III)

with a compound of formula (IV) or an activated derivative thereof

A suitable activated derivative of the compound of formula (IV) is theacid chloride. The reaction between the compound of formula (III) andcompound of formula (IV) may be carried out in a suitable organicsolvent optionally in the presence of a base.

Where the compound of formula (I) is a mixture of isomers the compoundsof formula (IA) and formula (IB) can be obtained from the compound offormula (I) using suitable separation techniques which are familiar tothose skilled in the art, such as those described herein. Alternativelythe compounds of formula (IA) and (IB) may be prepared by a chiralsynthesis procedure. By way of illustration, the compound of formula(IA) may be prepared by the procedure set out in Scheme 1.

It will be appreciated by those skilled in the art that it may beadvantageous to protect one or more functional groups of the compoundsdescribed above. Examples of protecting groups and the means for theirremoval can be found in T. W. Greene ‘Protective Groups in OrganicSynthesis’ (4th edition, J. Wiley and Sons, 2006). Suitable amineprotecting groups include acyl (e.g. acetyl, carbamate (e.g.2′,2′,2′-trichloroethoxycarbonyl, benzyloxycarbonyl or t-butoxycarbonyl)and arylalkyl (e.g. benzyl), which may be removed by hydrolysis (e.g.using an acid such as hydrochloric acid in dioxane or trifluoroaceticacid in dichloromethane) or reductively (e.g. hydrogenolysis of a benzylor benzyloxycarbonyl group or reductive removal of a2′,2′,2′-trichloroethoxycarbonyl group using zinc in acetic acid) asappropriate. Other suitable amine protecting groups includetrifluoroacetyl (—COCF₃) which may be removed by base catalysedhydrolysis.

It will be appreciated that in any of the routes described above, theprecise order of the synthetic steps by which the various groups andmoieties are introduced into the molecule may be varied. It will bewithin the skill of the practitioner in the art to ensure that groups ormoieties introduced at one stage of the process will not be affected bysubsequent transformations and reactions, and to select the order ofsynthetic steps accordingly.

Certain intermediate compounds described above form a yet further aspectof the invention.

The compounds of formula (I) and salts thereof are bromodomaininhibitors, and thus are believed to have potential utility in thetreatment of diseases or conditions for which a bromodomain inhibitor isindicated. Further, compounds of formula (I) and pharmaceuticallyacceptable salts thereof (such as the compound of formula (IA) or apharmaceutically acceptable salt thereof) may have one or more ADMET(absorption, distribution, metabolism, excretion and toxicity) propertythat makes it particularly suitable.

The present invention thus provides a compound of formula (I) or apharmaceutically acceptable salt thereof for use in therapy. In oneembodiment there is provided a compound of formula (IA) or apharmaceutically acceptable salt thereof for use in therapy.

The compound of formula (I) or a pharmaceutically acceptable saltthereof can be used in the treatment of diseases or conditions for whicha bromodomain inhibitor is indicated. The present invention thusprovides a compound of formula (I) or a pharmaceutically acceptable saltthereof for use in the treatment of any diseases or conditions for whicha bromodomain inhibitor is indicated. In one embodiment there isprovided a compound of formula (IA) or a pharmaceutically acceptablesalt thereof for use in the treatment of any diseases or conditions forwhich a bromodomain inhibitor is indicated. In another embodiment thereis provided a compound of formula (I) (such as a compound of formula(1A)) or a pharmaceutically acceptable salt thereof for use in thetreatment of chronic auto-immune and/or inflammatory conditions. In afurther embodiment there is provided a compound of formula (I) (such asa compound of formula (1A)) or a pharmaceutically acceptable saltthereof for use in the treatment of cancer.

Also provided is the use of a compound of formula (I) or apharmaceutically acceptable salt thereof in the manufacture of amedicament for the treatment of diseases or conditions for which abromodomain inhibitor is indicated. In one embodiment there is providedthe use of a compound of formula (IA) or a pharmaceutically acceptablesalt thereof in the manufacture of a medicament for the treatment ofdiseases or conditions for which a bromodomain inhibitor is indicated.

Also provided is a method of treating diseases or conditions for which abromodomain inhibitor is indicated in a subject in need thereof whichcomprises administering a therapeutically effective amount of compoundof formula (I) or a pharmaceutically acceptable salt thereof. In oneembodiment there is provided a method of treating diseases or conditionsfor which a bromodomain inhibitor is indicated in a subject in needthereof which comprises administering a therapeutically effective amountof a compound of formula (IA) or a pharmaceutically acceptable saltthereof.

Suitably the subject in need thereof is a mammal, particularly a human.

As used herein, the term “effective amount” means that amount of a drugor pharmaceutical agent that will elicit the biological or medicalresponse of a tissue, system, or subject (e.g. a human) that is beingsought, for instance, by a researcher or clinician. Furthermore, theterm “therapeutically effective amount” means any amount which, ascompared to a corresponding subject who has not received such amount,results in improved treatment, healing, prevention, or amelioration of adisease, disorder, or side effect, or a decrease in the rate ofadvancement of a disease or disorder. The term also includes within itsscope amounts effective to enhance normal physiological function.

Bromodomain inhibitors are believed to be useful in the treatment of avariety of diseases or conditions related to systemic or tissueinflammation, inflammatory responses to infection or hypoxia, cellularactivation and proliferation, lipid metabolism, fibrosis and in theprevention and treatment of viral infections.

Bromodomain inhibitors may be useful in the treatment of a wide varietyof chronic autoimmune and inflammatory conditions such as rheumatoidarthritis, osteoarthritis, acute gout, psoriasis, systemic lupuserythematosus, multiple sclerosis, inflammatory bowel disease (Crohn'sdisease and Ulcerative colitis), asthma, chronic obstructive airwaysdisease, pneumonitis, myocarditis, pericarditis, myositis, eczema,dermatitis (including atopic dermatitis), alopecia, vitiligo, bullousskin diseases, nephritis, vasculitis, atherosclerosis, Alzheimer'sdisease, depression, Sjögren's syndrome, sialoadenitis, central retinalvein occlusion, branched retinal vein occlusion, Irvine-Gass syndrome(post cataract and post-surgical), retinitis pigmentosa, pars planitis,birdshot retinochoroidopathy, epiretinal membrane, cystic macular edema,parafoveal telengiectasis, tractional maculopathies, vitreomaculartraction syndromes, retinal detachment, neuroretinitis, idiopathicmacular edema, retinitis, dry eye (kerartoconjunctivitis Sicca), vernalkeratoconjunctivitis, atopic keratoconjunctivitis, uveitis (such asanterior uveitis, pan uveitis, posterior uveits, uveitis-associatedmacula edema).scleritis, diabetic retinopathy, diabetic macula edema,age-related macula dystrophy, hepatitis, pancreatitis, primary biliarycirrhosis, sclerosing cholangitis, Addison's disease, hypophysitis,thyroiditis, type I diabetes and acute rejection of transplanted organs.

Bromodomain inhibitors may be useful in the treatment of a wide varietyof acute inflammatory conditions such as acute gout, giant cellarteritis, nephritis including lupus nephritis, vasculitis with organinvolvement such as glomerulonephritis, vasculitis including giant cellarteritis, Wegener's granulomatosis, Polyarteritis nodosa, Behcet'sdisease, Kawasaki disease, Takayasu's Arteritis, pyoderma gangrenosum,vasculitis with organ involvement and acute rejection of transplantedorgans.

Bromodomain inhibitors may be useful in the treatment of diseases orconditions which involve inflammatory responses to infections withbacteria, viruses, fungi, parasites or their toxins, such as sepsis,sepsis syndrome, septic shock, endotoxaemia, systemic inflammatoryresponse syndrome (SIRS), multi-organ dysfunction syndrome, toxic shocksyndrome, acute lung injury, ARDS (adult respiratory distress syndrome),acute renal failure, fulminant hepatitis, burns, acute pancreatitis,post-surgical syndromes, sarcoidosis, Herxheimer reactions,encephalitis, myelitis, meningitis, malaria and SIRS associated withviral infections such as influenza, herpes zoster, herpes simplex andcoronavirus.

Bromodomain inhibitors may be useful in the treatment of conditionsassociated with ischaemia-reperfusion injury such as myocardialinfarction, cerebro-vascular ischaemia (stroke), acute coronarysyndromes, renal reperfusion injury, organ transplantation, coronaryartery bypass grafting, cardio-pulmonary bypass procedures, pulmonary,renal, hepatic, gastro-intestinal or peripheral limb embolism.

Bromodomain inhibitors may be useful in the treatment of disorders oflipid metabolism via the regulation of APO-A1 such ashypercholesterolemia, atherosclerosis and Alzheimer's disease.

Bromodomain inhibitors may be useful in the treatment of fibroticconditions such as idiopathic pulmonary fibrosis, renal fibrosis,post-operative stricture, keloid scar formation, scleroderma (includingmorphea) and cardiac fibrosis.

Bromodomain inhibitors may be useful in the treatment of viralinfections such as herpes virus, human papilloma virus, adenovirus andpoxvirus and other DNA viruses.

Bromodomain inhibitors may be useful in the treatment of cancer,including hematological (such as leukaemia, lymphoma and multiplemyeloma), epithelial including lung, breast and colon carcinomas,midline carcinomas, mesenchymal, hepatic, renal and neurologicaltumours.

Bromodomain inhibitors may be useful in the treatment of one or morecancers selected from brain cancer (gliomas), glioblastomas,Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease,breast cancer, inflammatory breast cancer, colorectal cancer, Wilm'stumor, Ewing's sarcoma, rhabdomyosarcoma, ependymoma, medulloblastoma,colon cancer, head and neck cancer, kidney cancer, lung cancer, livercancer, melanoma, squamous cell carcinoma, ovarian cancer, pancreaticcancer, prostate cancer, sarcoma cancer, osteosarcoma, giant cell tumorof bone, thyroid cancer, lymphoblastic T-cell leukemia, chronicmyelogenous leukemia, chronic lymphocytic leukemia, hairy-cell leukemia,acute lymphoblastic leukemia, acute myelogenous leukemia, chronicneutrophilic leukemia, acute lymphoblastic T-cell leukemia,plasmacytoma, immunoblastic large cell leukemia, mantle cell leukemia,multiple myeloma, megakaryoblastic leukemia, acute megakaryocyticleukemia, promyelocytic leukemia, mixed lineage leukaemia,erythroleukemia, malignant lymphoma, Hodgkins lymphoma, non-Hodgkinslymphoma, lymphoblastic T-cell lymphoma, Burkitt's lymphoma, follicularlymphoma, neuroblastoma, bladder cancer, urothelial cancer, vulvalcancer, cervical cancer, endometrial cancer, renal cancer, mesothelioma,esophageal cancer, salivary gland cancer, hepatocellular cancer, gastriccancer, nasopharangeal cancer, buccal cancer, cancer of the mouth, GIST(gastrointestinal stromal tumor) and testicular cancer.

In one embodiment the cancer is a leukaemia, for example a leukaemiaselected from acute monocytic leukemia, acute myelogenous leukemia,chronic myelogenous leukemia, chronic lymphocytic leukemia and mixedlineage leukaemia (MLL), In another embodiment the cancer is NUT-midlinecarcinoma. In another embodiment the cancer is multiple myeloma. Inanother embodiment the cancer is a lung cancer such as small cell lungcancer (SCLC). In another embodiment the cancer is a neuroblastoma. Inanother embodiment the cancer is Burkitt's lymphoma. In anotherembodiment the cancer is cervical cancer. In another embodiment thecancer is esophageal cancer. In another embodiment the cancer is ovariancancer. In another embodiment the cancer is breast cancer. In anotherembodiment the cancer is colarectal cancer.

In one embodiment the disease or condition for which a bromodomaininhibitor is indicated is selected from diseases associated withsystemic inflammatory response syndrome, such as sepsis, burns,pancreatitis, major trauma, haemorrhage and ischaemia. In thisembodiment the bromodomain inhibitor would be administered at the pointof diagnosis to reduce the incidence of: SIRS, the onset of shock,multi-organ dysfunction syndrome, which includes the onset of acute lunginjury, ARDS, acute renal, hepatic, cardiac or gastro-intestinal injuryand mortality. In another embodiment the bromodomain inhibitor would beadministered prior to surgical or other procedures associated with ahigh risk of sepsis, haemorrhage, extensive tissue damage, SIRS or MODS(multiple organ dysfunction syndrome). In a particular embodiment thedisease or condition for which a bromodomain inhibitor is indicated issepsis, sepsis syndrome, septic shock and endotoxaemia. In anotherembodiment, the bromodomain inhibitor is indicated for the treatment ofacute or chronic pancreatitis. In another embodiment the bromodomain isindicated for the treatment of burns.

In one embodiment the disease or condition for which a bromodomaininhibitor is indicated is selected from herpes simplex infections andreactivations, cold sores, herpes zoster infections and reactivations,chickenpox, shingles, human papilloma virus, human immunodeficiencyvirus (HIV), cervical neoplasia, adenovirus infections, including acuterespiratory disease, poxvirus infections such as cowpox and smallpox andAfrican swine fever virus. In one particular embodiment a bromodomaininhibitor is indicated for the treatment of Human papilloma virusinfections of skin or cervical epithelia. In one embodiment thebromodomain inhibitor is indicated for the treatment of latent HIVinfection.

As used herein the reference to the “treatment” of a particular diseaseor condition includes the prevention or prophylaxis of such a disease orcondition.

The term “diseases or conditions for which a bromodomain inhibitor isindicated”, is intended to include each of or all of the above diseasesor conditions.

The invention further provides for a method for inhibiting a bromodomainwhich comprises contacting the bromodomain with a compound of formula(I) or a pharmaceutically acceptable salt thereof.

While it is possible that for use in therapy, a compound of formula (I)as well as pharmaceutically acceptable salts thereof may be administeredas the raw chemical, it is common to present the active ingredient as apharmaceutical composition.

The present invention therefore provides in a further aspect apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt and one or more pharmaceuticallyacceptable carriers, diluents or excipients. In one embodiment there isprovided a pharmaceutical composition comprising a compound of formula(IA) or a pharmaceutically acceptable salt and one or morepharmaceutically acceptable carriers, diluents or excipients. Thecompounds of formula (I) and pharmaceutically acceptable salts, are asdescribed above. The carrier(s), diluent(s) or excipient(s) must beacceptable in the sense of being compatible with the other ingredientsof the composition and not deleterious to the recipient thereof. Inaccordance with another aspect of the invention there is also provided aprocess for the preparation of a pharmaceutical composition includingadmixing a compound of formula (I), or a pharmaceutically acceptablesalt thereof, with one or more pharmaceutically acceptable carriers,diluents or excipients. The pharmaceutical composition can be used inthe treatment of any of the conditions described herein.

Since the compounds of formula (I) are intended for use inpharmaceutical compositions it will be readily understood that they areeach preferably provided in substantially pure form, for example, atleast 85% pure, especially at least 98% pure (% in a weight for weightbasis).

Pharmaceutical compositions may be presented in unit dose formscontaining a predetermined amount of active ingredient per unit dose.Preferred unit dosage compositions are those containing a daily dose orsub-dose, or an appropriate fraction thereof, of an active ingredient.Such unit doses may therefore be administered more than once a day.Preferred unit dosage compositions are those containing a daily dose orsub-dose (for administration more than once a day), as herein aboverecited, or an appropriate fraction thereof, of an active ingredient.

Pharmaceutical compositions may be adapted for administration by anyappropriate route, for example by the oral (including buccal orsublingual), rectal, inhaled, intranasal, topical (including buccal,sublingual or transdermal), ocular (including topical, intraocular,subconjunctival, episcleral, sub-Tenon), vaginal or parenteral(including subcutaneous, intramuscular, intravenous or intradermal)route. Such compositions may be prepared by any method known in the artof pharmacy, for example by bringing into association the activeingredient with the carrier(s) or excipient(s).

In one embodiment the pharmaceutical composition is adapted forparenteral administration, particularly intravenous administration.

In one embodiment the pharmaceutical composition is adapted for oraladministration.

In one embodiment the pharmaceutical composition is adapted for topicaladministration.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe composition isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The compositions may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets.

Pharmaceutical compositions adapted for oral administration may bepresented as discrete units such as capsules or tablets; powders orgranules; solutions or suspensions in aqueous or non-aqueous liquids;edible foams or whips; or oil-in-water liquid emulsions or water-in-oilliquid emulsions.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Powders suitable for incorporating intotablets or capsules may be prepared by reducing the compound to asuitable fine size (e.g. by micronisation) and mixing with a similarlyprepared pharmaceutical carrier such as an edible carbohydrate, forexample, starch or mannitol. Flavoring, preservative, dispersing andcoloring agent can also be present.

Capsules may be made by preparing a powder mixture, as described above,and filling formed gelatin sheaths. Glidants and lubricants such ascolloidal silica, talc, magnesium stearate, calcium stearate or solidpolyethylene glycol can be added to the powder mixture before thefilling operation. A disintegrating or solubilizing agent such asagar-agar, calcium carbonate or sodium carbonate can also be added toimprove the availability of the medicament when the capsule is ingested.

Moreover, when desired or necessary, suitable binders, glidants,lubricants, sweetening agents, flavours, disintegrating agents andcoloring agents can also be incorporated into the mixture. Suitablebinders include starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include sodium oleate, sodium stearate, magnesium stearate, sodiumbenzoate, sodium acetate, sodium chloride and the like. Disintegratorsincludestarch, methyl cellulose, agar, bentonite, xanthan gum and thelike. Tablets are formulated, for example, by preparing a powdermixture, granulating or slugging, adding a lubricant and disintegrantand pressing into tablets. A powder mixture is prepared by mixing thecompound, suitably comminuted, with a diluent or base as describedabove, and optionally, with a binder such as carboxymethylcellulose, analiginate, gelatin, or polyvinyl pyrrolidone, a solution retardant suchas paraffin, a resorption accelerator such as a quaternary salt and/oran absorption agent such as bentonite, kaolin or dicalcium phosphate.The powder mixture can be granulated by wetting with a binder such assyrup, starch paste, acadia mucilage or solutions of cellulosic orpolymeric materials and forcing through a screen. As an alternative togranulating, the powder mixture can be run through the tablet machineand the result is imperfectly formed slugs broken into granules. Thegranules can be lubricated to prevent sticking to the tablet formingdies by means of the addition of stearic acid, a stearate salt, talc ormineral oil. The lubricated mixture is then compressed into tablets. Thecompounds of formula (I) and pharmaceutically acceptable salts thereof,can also be combined with a free flowing inert carrier and compressedinto tablets directly without going through the granulating or sluggingsteps. A clear or opaque protective coating consisting of a sealing coatof shellac, a coating of sugar or polymeric material and a polishcoating of wax can be provided. Dyestuffs can be added to these coatingsto distinguish different unit dosages.

Oral fluids such as solution, syrups and elixirs can be prepared indosage unit form so that a given quantity contains a predeterminedamount of the compound. Syrups can be prepared by dissolving thecompound in a suitably flavored aqueous solution, while elixirs areprepared through the use of a non-toxic alcoholic vehicle. Suspensionscan be formulated by dispersing the compound in a non-toxic vehicle.Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols andpolyoxy ethylene sorbitol ethers, preservatives, flavor additive such aspeppermint oil or natural sweeteners or saccharin or other artificialsweeteners, and the like can also be added.

Compositions for administration (e.g. oral administration) may bedesigned to provide a modified release profile so as to sustain orotherwise control the release of the therapeutically active agent. Amodified release profile of the therapeutically active agent may beobtained through the design of polymeric matrices incorporatingdifferent choices and properties of biodegradable/bioerodable polymers(e.g. poly(ethylene vinyl) acetate (EVA), superhydrolyzed PVA),hydroxyalkyl cellulose (HPC), methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), polycaprolactone, poly(glycolic) acid, poly(lactic)acid, polyanhydride, of polymer molecular weights, polymercrystallinity, copolymer ratios, processing conditions, surface finish,geometry, excipient addition and polymeric coatings that will enhancedrug diffusion, erosion, dissolution and osmosis.

Where appropriate, dosage unit compositions for oral administration canbe microencapsulated. The composition may be prepared to prolong orsustain the release as for example by coating or embedding particulatematerial in polymers, wax or the like.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof, can also be administered in the form of liposome deliverysystems, such as small unilamellar vesicles, large unilamellar vesiclesand multilamellar vesicles. Liposomes can be formed from a variety ofphospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, emulsions, lotions,powders, solutions, pastes, gels, foams, sprays, aerosols or oils. Suchpharmaceutical compositions may include conventional additives whichinclude, but are not limited to, preservatives, solvents to assist drugpenetration, co-solvents, emollients, propellants, viscosity modifyingagents (gelling agents), surfactants and carriers. In one embodimentthere is provided a pharmaceutical composition adapted for topicaladministration which comprises between 0.01-10%, or between 0.01-1% ofthe compound of formula (I), or a pharmaceutically acceptable saltthereof, by weight of the composition.

For treatments of the eye or other external tissues, for example mouthand skin, the compositions are preferably applied as a topical ointment,cream, gel, spray or foam. When formulated in an ointment, the activeingredient may be employed with either a paraffinic or a water-miscibleointment base. Alternatively, the active ingredient may be formulated ina cream with an oil-in-water cream base or a water-in-oil base.

Pharmaceutical compositions adapted for topical administrations to theeye include eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent.Compositions to be administered to the eye will have ophthalmicallycompatible pH and osmolality. One or more ophthalmically acceptable pHadjusting agents and/or buffering agents can be included in acomposition of the invention, including acids such as acetic, boric,citric, lactic, phosphoric and hydrochloric acids; bases such as sodiumhydroxide, sodium phosphate, sodium borate, sodium citrate, sodiumacetate, and sodium lactate; and buffers such as citrate/dextrose,sodium bicarbonate and ammonium chloride. Such acids, bases, and bufferscan be included in an amount required to maintain pH of the compositionin an ophthalmically acceptable range. One or more ophthalmicallyacceptable salts can be included in the composition in an amountsufficient to bring osmolality of the composition into an ophthalmicallyacceptable range. Such salts include those having sodium, potassium orammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions.

The ocular delivery device may be designed for the controlled release ofone or more therapeutic agents with multiple defined release rates andsustained dose kinetics and permeability.

Pharmaceutical compositions for ocular delivery also include in situgellable aqueous composition. Such a composition comprises a gellingagent in a concentration effective to promote gelling upon contact withthe eye or with lacrimal fluid. Suitable gelling agents include but arenot limited to thermosetting polymers. The term “in situ gellable” asused herein is includes not only liquids of low viscosity that form gelsupon contact with the eye or with lacrimal fluid, but also includes moreviscous liquids such as semi-fluid and thixotropic gels that exhibitsubstantially increased viscosity or gel stiffness upon administrationto the eye. See, for example, Ludwig (2005) Adv. Drug Deliv. Rev. 3;57:1595-639, herein incorporated by reference for purposes of itsteachings of examples of polymers for use in ocular drug delivery.

Dosage forms for nasal or inhaled administration may conveniently beformulated as aerosols, solutions, suspensions, gels or dry powders.

For compositions suitable and/or adapted for inhaled administration, itis preferred that the compound of formula (I) or a pharmaceuticallyacceptable salt thereof, is in a particle-size-reduced form e.g.obtained by micronisation. The preferable particle size of thesize-reduced (e.g. micronised) compound or salt is defined by a D50value of about 0.5 to about 10 microns (for example as measured usinglaser diffraction).

Aerosol formulations, e.g. for inhaled administration, can comprise asolution or fine suspension of the active substance in apharmaceutically acceptable aqueous or non-aqueous solvent. Aerosolformulations can be presented in single or multidose quantities insterile form in a sealed container, which can take the form of acartridge or refill for use with an atomising device or inhaler.Alternatively the sealed container may be a unitary dispensing devicesuch as a single dose nasal inhaler or an aerosol dispenser fitted witha metering valve (metered dose inhaler) which is intended for disposalonce the contents of the container have been exhausted.

Where the dosage form comprises an aerosol dispenser, it preferablycontains a suitable propellant under pressure such as compressed air,carbon dioxide or an organic propellant such as a hydrofluorocarbon(HFC). Suitable HFC propellants include 1,1,1,2,3,3,3-heptafluoropropaneand 1,1,1,2-tetrafluoroethane. The aerosol dosage forms can also takethe form of a pump-atomiser. The pressurised aerosol may contain asolution or a suspension of the active compound. This may require theincorporation of additional excipients e.g. co-solvents and/orsurfactants to improve the dispersion characteristics and homogeneity ofsuspension formulations. Solution formulations may also require theaddition of co-solvents such as ethanol.

For pharmaceutical compositions suitable and/or adapted for inhaledadministration, the pharmaceutical composition may be a dry powderinhalable composition. Such a composition can comprise a powder basesuch as lactose, glucose, trehalose, mannitol or starch, the compound offormula (I) or a pharmaceutically acceptable salt thereof (preferably inparticle-size-reduced form, e.g. in micronised form), and optionally aperformance modifier such as L-leucine or another amino acid and/ormetal salt of stearic acid such as magnesium or calcium stearate.Preferably, the dry powder inhalable composition comprises a dry powderblend of lactose e.g. lactose monohydrate and the compound of formula(I) or salt thereof. Such compositions can be administered to thepatient using a suitable device such as the DISKUS® device, marketed byGlaxoSmithKline which is for example described in GB 2242134 A.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may be formulated as a fluid formulation for delivery from afluid dispenser, for example a fluid dispenser having a dispensingnozzle or dispensing orifice through which a metered dose of the fluidformulation is dispensed upon the application of a user-applied force toa pump mechanism of the fluid dispenser. Such fluid dispensers aregenerally provided with a reservoir of multiple metered doses of thefluid formulation, the doses being dispensable upon sequential pumpactuations. The dispensing nozzle or orifice may be configured forinsertion into the nostrils of the user for spray dispensing of thefluid formulation into the nasal cavity. A fluid dispenser of theaforementioned type is described and illustrated in WO-A-2005/044354.

A therapeutically effective amount of a compound of formula (I) or apharmaceutically acceptable salt thereof, will depend upon a number offactors including, for example, the age and weight of the subject, theprecise condition requiring treatment and its severity, the nature ofthe formulation, and the route of administration, and will ultimately beat the discretion of the attendant physician or veterinarian. In thepharmaceutical composition, each dosage unit for oral or parenteraladministration preferably contains from 0.01 to 3000 mg, more preferably0.5 to 1000 mg, of a compound of formula (I) or a pharmaceuticallyacceptable salt thereof, calculated as the free base. Each dosage unitfor nasal or inhaled administration preferably contains from 0.001 to 50mg, more preferably 0.01 to 5 mg, of a compound of the formula (I) or apharmaceutically acceptable salt thereof, calculated as the free base.

The pharmaceutically acceptable compounds of formula (I) andpharmaceutically acceptable salts thereof, can be administered in adaily dose (for an adult patient) of, for example, an oral or parenteraldose of 0.01 mg to 3000 mg per day, 0.5 to 1000 mg per day or 100 mg to2500 mg per day, or a nasal or inhaled dose of 0.001 to 50 mg per day or0.01 to 5 mg per day, of the compound of the formula (I) or apharmaceutically acceptable salt thereof, calculated as the free base.This amount may be given in a single dose per day or more usually in anumber (such as two, three, four, five or six) of sub-doses per day suchthat the total daily dose is the same. An effective amount of a saltthereof, may be determined as a proportion of the effective amount ofthe compound of formula (I) per se.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof, and may be employed alone or in combination with othertherapeutic agents. Combination therapies according to the presentinvention thus comprise the administration of at least one compound offormula (I) or a pharmaceutically acceptable salt thereof, and the useof at least one other therapeutically active agent. Preferably,combination therapies according to the present invention comprise theadministration of at least one compound of formula (I) or apharmaceutically acceptable salt thereof, and at least one othertherapeutically active agent. The compound(s) of formula (I) andpharmaceutically acceptable salts thereof, and the other therapeuticallyactive agent(s) may be administered together in a single pharmaceuticalcomposition or separately and, when administered separately this mayoccur simultaneously or sequentially in any order. The amounts of thecompound(s) of formula (I) and pharmaceutically acceptable saltsthereof, and the other therapeutically active agent(s) and the relativetimings of administration will be selected in order to achieve thedesired combined therapeutic effect. Thus in a further aspect, there isprovided a combination comprising a compound of formula (I) or apharmaceutically acceptable salt thereof, and at least one othertherapeutically active agent.

Thus in one aspect, the compound of formula (I) or a pharmaceuticallyacceptable salt thereof, and pharmaceutical compositions comprising acompound of formula (I) or a pharmaceutically acceptable salt thereof,according to the invention may be used in combination with or includeone or more other therapeutic agents, for example selected fromantibiotics, anti-virals, glucocorticosteroids, muscarinic antagonistsbeta-2 agonists and Vitamin D3 analogues. In a further embodiment acompound of formula (I) or a pharmaceutically acceptable salt thereofmay be used in combination with a further therapeutic agent which issuitable for the treatment of cancer. Examples of such furthertherapeutic agents are desfibed in Cancer Principles and Practice ofOncology by V. T. Devita and S. Hellman (editors), 6^(th) edition(2001), Lippincott Williams & Wilkins Publishers. A person of ordinaryskill in the art would be able to discern which combinations of agentswould be useful based on the particular characteristics of the drugs andthe cancer involved. Further therapeutic agents to be used incombination with the compound of formula (I) or a pharmaceuticallyacceptable salt thereof include, but are not limited to,anti-microtubule agents (such as diterpenoids and vinca alkaloids);platinum coordination complexes; alkylating agents (such as nitrogenmustards, oxazaphosphorines, alkylsulfonates, nitrosoureas, andtriazenes); antibiotic agents (such as anthracyclins, actinomycins andbleomycins); topoisomerase II inhibitors (such as epipodophyllotoxins);antimetabolites (such as purine and pyrimidine analogues and anti-folatecompounds); topoisomerase I inhibitors (such as camptothecins; hormonesand hormonal analogues); signal transduction pathway inhibitors (such astyropsine receptor inhibitors); non-receptor tyrosine kinaseangiogenesis inhibitors; immunotherapeutic agents; proapoptotic agents;epigenetic or transcriptional modulators (such as histone deacetylaseinhibitors) and cell cycle signaling inhibitors.

It will be appreciated that when the compound of formula (I) or apharmaceutically acceptable salt thereof, is administered in combinationwith other therapeutic agents normally administered by the inhaled,intravenous, oral or intranasal route, that the resultant pharmaceuticalcomposition may be administered by the same routes. Alternatively theindividual components of the composition may be administered bydifferent routes.

One embodiment of the invention encompasses combinations comprising oneor two other therapeutic agents.

It will be clear to a person skilled in the art that, where appropriate,the other therapeutic ingredient(s) may be used in the form of salts,for example as alkali metal or amine salts or as acid addition salts, orprodrugs, or as esters, for example lower alkyl esters, or as solvates,for example hydrates, to optimise the activity and/or stability and/orphysical characteristics, such as solubility, of the therapeuticingredient. It will be clear also that, where appropriate, thetherapeutic ingredients may be used in optically pure form.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical composition and thus pharmaceuticalcompositions comprising a combination as defined above together with apharmaceutically acceptable diluent or carrier represent a furtheraspect of the invention.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof, may be prepared by the methods described below or by similarmethods. Thus the following Intermediates and Examples serve toillustrate the preparation of the compounds of formula (I) andpharmaceutically acceptable salts thereof, and are not to be consideredas limiting the scope of the invention in any way.

General Experimental Details

All temperatures referred to are in ° C.

The names of the following compounds have been obtained using thecompound naming programme “ACD Name Pro 6.02” or Chem Draw Ultra 12.0.

Abbreviations

-   1,2-DCE 1,2-dichloroethane-   AcOH acetic acid-   CHCl₃ chloroform-   D6-DMSO deuterated dimethylsulfoxide-   DCM dichloromethane-   DIPEA diisopropylamine-   DMSO dimethylsulfoxide-   DPPA diphenylphosphoryl azide-   Et₃N triethylamine-   EtOAc ethyl acetate-   h hour(s)-   HATU O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate-   HCl hydrochloric acid-   i-PrOac isopropylacetate-   i-Pr₂O diisopropyl ether-   LCMS liquid chromatography-mass spectrometry-   LiOH lithium hydroxide-   M molar (concentration)-   MeCN acetonitrile-   MeOH methanol-   min minute(s)-   N normal (concentration)-   Na₂CO₃ sodium carbonate-   Na₂SO₄ sodium sulphate-   Pd/C palladium on carbon-   Rt retention time-   TBME tertiary butyl methyl ether-   TFA trifluoroacetic acid-   THF tetrahydrofuran-   UPLC Ultra performance liquid chromatograpy

LCMS Methodology Method Formate LC Conditions

The UPLC analysis was conducted on an Acquity UPLC BEH C18 column (50mm×2.1 mm, i.d. 1.7 μm packing diameter) at 40° C.

The solvents employed were:

A=0.1% v/v solution of formic acid in water

B=0.1% v/v solution of formic acid in acetonitrile

The gradient employed was:

Time (min) Flow rate (ml/min) % A % B 0 1 97 3 1.5 1 0 100 1.9 1 0 1002.0 1 97 3

The UV detection was a summed signal from wavelength of 210 nm to 350nm.

MS conditions

MS: Waters ZQ

Ionisation mode: Alternate-scan positive and negative electrosprayScan range: 100 to 1000 AMUScan time: 0.27 secInter scan: delay 0.10 sec

NMR

Spectra were run on a 400 mHz NMR machine at either 302 K or for VTspectra at 392-393 K.

Intermediate 17-(3,5-dimethylisoxazol-4-yl)-6-methoxy-N-(1-methoxypropan-2-yl)-3-nitroquinolin-4-amine

To a solution of4-(4-chloro-6-methoxy-3-nitroquinolin-7-yl)-3,5-dimethylisoxazole(Manchester Organics) (20 g, 59.9 mmol) in 1,4-dioxane (200 ml) wasadded 1-methoxypropan-2-amine (31.6 ml, 300 mmol) and the reactionmixture heated at 70° C. for 1.5 h. The solvent was removed underreduced pressure and the resulting solid partitioned between ethylacetate (3×750 ml) and water (750 ml). The organic layers were combined,dried (hydrophobic frit) and evaporated under reduced pressure to give7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-N-(1-methoxypropan-2-yl)-3-nitroquinolin-4-amine (25.8 g), which was used without further purification inthe subsequent step.

NMR (D₆-DMSO): OH 9.03 (s, 1H), 8.63 (d, 1H), 7.83 (s, 1H), 7.81 (s,1H), 4.39 (m, 1H), 3.97 (s, 3H), 3.54 (m, 2H), 3.27 (s, 3H), 2.34 (s,3H), 2.15 (s, 3H), 1.41 (d, 3H).

LCMS (formate): Rt=0.97 min, MH⁺387.

Intermediate 27-(3,5-dimethylisoxazol-4-yl)-6-methoxy-N4-(1-methoxypropan-2-yl)quinoline-3,4-diamine

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-N-(1-methoxypropan-2-yl)-3-nitroquinolin-4-amine(for a preparation see Intermediate 1)(25.8 g, 66.8 mmol) was dissolvedin a mixture of ethyl acetate (1000 ml) and DMSO (50 ml) and thesolution was hydrogenated using a flow-hydrogenation apparatus (H-cube™)(settings: 20° C., 1 bar, 1 ml/min flow rate) and a 10% Pd/C CatCart 70catalyst cartridge. The catalyst cartridge was changed whenever itbecame blocked. The reaction mixture was evaporated under reducedpressure and partitioned between ethyl acetate (750 ml) and water (3×750ml). The aqueous layers were combined and extracted with ethyl acetate(750 ml). The organic layers were were combined, dried (hydrophobicfrit) and evaporated under reduced pressure. The residue was dissolvedin DCM and applied to a silica cartridge (100 g). The cartridge waseluted with a 2M ammonia in methanol/DCM gradient (0-4%). Theappropriate fractions were combined and evaporated under reducedpressure to give7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-N⁴-(1-methoxypropan-2-yl)quinoline-3,4-diamineand recovered7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-N-(1-methoxypropan-2-yl)-3-nitroquinolin-4-amine(6.5 g)

The recovered starting material was dissolved in ethyl acetate (250 ml)and the solution was hydrogenated using a flow-hydrogenation apparatus(H-cube™) (settings: 20° C., 1 bar, 1 ml/min flow rate) and 10% Pd/CCatCart 70 catalyst cartridge. The reaction mixture was evaporated underreduced pressure and the residue was dissolved in DCM and applied to asilica cartridge (100 g). The cartridge was eluted with a 2M ammonia inmethanol/DCM gradient (0-4%). The appropriate fractions were combinedand evaporated under reduced pressure to give7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-N4-(1-methoxypropan-2-yl)quinoline-3,4-diamine

The batches of product were combined to give7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-N4-(1-methoxypropan-2-yl)quinoline-3,4-diamine(15.6 g, 43.8 mmol, 65.6% yield) as a sticky dark brown gum.

NMR (D₆-DMSO): δH 8.29 (s, 1H), 7.55 (s, 1H), 7.45 (s, 1H), 5.13 (s,2H), 4.48 (d, 1H), 3.88 (s, 3H), 3.54 (m, 1H), 3.35 (m, 2H), 3.29 (s,3H), 2.30 (s, 3H), 2.10 (s, 3H), 1.18 (d, 3H). LCMS (formate): Rt 0.73min, MH⁺357

Intermediate 3N-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-4-((1-methoxypropan-2-yl)amino)quinolin-3-yl)tetrahydro-2H-pyran-4-carboxamide

A solution of7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-N4-(1-methoxypropan-2-yl)quinoline-3,4-diamine(for a preparation see Intermediate 2) (9.1 g) in DCM (300 ml) wastreated with pyridine (30 ml) and tetrahydro-2H-pyran-4-carbonylchloride (5.0 ml) and the solution stirred under nitrogen at ambienttemperature for 3.5 h and then left standing overnight (ambienttemperature, under nitrogen). The volatiles were evaporated in vacuo andthe residue partitioned between DCM and water. The organic phase waswashed with water ×2 and the combined aqueous extracted with DCM. Thecombined organic phases were washed with brine, dried (hydrophobic frit)and reduced to dryness in vacuo. The combined aqueous including thebrine wash (˜pH4) was basified with solid sodium hydrogen carbonate andthe aqueous extracted with DCM x2. The organic fractions were dried(hydrophobic frit), combined with the previous material and reduced todryness in vacuo to give a brown foam. This foam was further dried invacuo, triturated with diethyl ether, the suspension chilled (ice/waterbath) and the solid isolated by filtration. The solid was washed with alittle diethyl ether and air-dried to giveN-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-4-((1-methoxypropan-2-yl)amino)quinolin-3-yl)tetrahydro-2H-pyran-4-carboxamideas a beige solid (11.95 g, 100%).

NMR (D₆-DMSO): δH 9.49 (s, 1H), 8.38 (s, 1H), 7.70 (s, 1H), 7.63 (s,1H), 5.33 (d, 1H), 3.96-3.90 (m, 6H), 3.43-3.28 (m partially obscured bywater, 7H), 2.69 (m, 1H), 2.32 (s, 3H), 2.12 (s, 3H), 1.81-1.67 (m, 4H),1.19 (d, 3H). LCMS (formate): Rt 0.69 mins, MH⁺469.

Example 14-(8-methoxy-1-(1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole

A suspension ofN-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-4-((1-methoxypropan-2-yl)amino)quinolin-3-yl)tetrahydro-2H-pyran-4-carboxamide(for a preparation see Intermediate 3) (29.4 g, 62.7 mmol) in aceticacid (250 ml, 62.7 mmol) was heated at 120° C. for 2 h. 3 Å molecularsieves (20 g) were added and heating continued for 3.5 h. Further 3 Åmolecular sieves (20 g, oven dried) were added and heating was continuedovernight. Further 3 Å molecular sieves (20 g, oven dried) were addedand heating continued for a further 24 h.

The reaction mixture was allowed to cool to room temperature, and thesolid removed by filtration. The residue and filtrate were combined andevaporated under reduced pressure. Water (3 l) was added, and theresultant slurry was neutralised by the slow addition of solid sodiumhydrogen carbonate. The aqueous slurry was extracted with DCM (3×1 l),and the organic phases were combined, dried (hydrophobic frit) andevaporated under reduced pressure to give a brown gum.

The gum was dissolved with warming and sonication in a minimal quantityof DCM. The solution was applied to a silica column (750 g) which hadbeen pre-wetted with DCM. The column was eluted with a gradient of [2 Mammonia in methanol, methanol (3:1)]/DCM (0-˜8%). The product fractionswere combined and reduced to dryness under reduced pressure to give acream foam/glass (13.027 g).

The mixed product fractions were combined and reduced to dryness underreduced pressure to give a deep yellow oil. This oil was dissolved indiethyl ether and the volatiles evaporated in vacuo to give a yellowsolid. The solid was triturated with diethyl ether, the solid isolatedby filtration and washed with diethyl ether (×2) to give4-(8-methoxy-1-(1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole.

The cream foam/glass was triturated with diethyl ether/ethyl acetate,the mixture reduced to dryness under reduced pressure and thetrituration was repeated with diethyl ether. The previous productfraction was added to the suspension and the mixture aged overnight. Thesolid was isolated by filtration and washed with diethyl ether to give4-(8-methoxy-1-(1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazoleas a cream solid. The solid was dried in vacuo and retriturated withdiethyl ether with stirring over ˜30 min. The solid was isolated byfiltration and washed with diethyl ether. The solid was dried in vacuoto give4-(8-methoxy-1-(1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazoleas a white solid (11.73 g).

VT NMR (D₆-DMSO): δH 9.04 (s, 1H), 7.99 (s, 1H), 7.74 (s, 1H), 5.45 (m,1H), 4.14 (m, 1H), 4.06-4.01 (m, 6H), 3.62 (m, 2H), 3.47 (m, 1H), 3.28(s, 3H), 2.35 (s, 3H), 2.17 (s, 3H), 2.09 (m, 2H), 1.92 (m, 2H), 1.83(d, 3H). LCMS (formate): Rt 0.76 min, MH⁺451.

Example 1 Alternative Preparation4-(8-methoxy-1-(1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole

A mixture ofN-(7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-4-((1-methoxypropan-2-yl)amino)quinolin-3-yl)tetrahydro-2H-pyran-4-carboxamide(for a preparation see Intermediate 3) (11.95 g, 25.5 mmol) andp-toluenesulfonic acid (1.2 g, 25.5 mmol) in toluene (250 ml) was heatedunder nitrogen at reflux using a Dean-Stark apparatus for 3 days.Further p-toluenesulfonic acid (0.2 g, 4.3 mmol) was added and heatingcontinued overnight. Water (750 ml) was added, and the mixture basifiedto pH 8 using saturated aqueous sodium hydrogen carbonate solution. Theaqueous was extracted with ethyl acetate (3×750 ml), the organic layerscombined, dried (hydrophobic frit) and evaporated under reducedpressure. The residual solid was triturated in ether (˜200 ml),sonicated briefly. The majority of the solid appeared to be a finepowder. The fine powder suspended in the ether was decanted, the solidisolated by filtration, and dried in a vacuum oven to give crude4-(8-methoxy-1-(1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(6.3 g). This material was triturated in ether (˜100 ml), sonicatedbriefly, and stood overnight at room temperature. The solid was isolatedby filtration and dried in a vacuum oven4-(8-methoxy-1-(1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(4.9 g, 10.88 mmol, 42.6% yield). LCMS (formate): Rt 0.75 min, MH⁺451.

The clumped solid left over from the decanting was triturated in ether(100 ml), sonicated for 15 min, and stood at room temperature for 3days. The solid was isolated by filtration and dried in a vacuum oven togive4-(8-methoxy-1-(1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(3.6 g, 7.99 mmol, 31.3% yield). LCMS (formate): Rt 0.76 min, MH⁺451.

Example 24-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole

Chiral resolution of4-(8-methoxy-1-(1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazolewas carried out using the following conditions:

Column: Chiralpak AD-H (250×30 mm, 5 micron) [ADH10029-01]Flowrate: 45 ml/minDetection: UV DAD (300 nm (bandwidth 180 nm, reference 550 nm (bandwidth100 nm)).Mobile Phase A: n-Hexane (10 ml of isopropylamine per Winchester (2.5L))Mobile Phase B: Ethanol (10 ml of isopropylamine per Winchester (2.5 L))Isocratic system—85:15 mobile phase A:BRun time—ca. 35 min

4-(8-methoxy-1-(1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(500 mg) was suspended in ethanol and ethylene glycol (10 ml:5 ml) andthen sonicated and heated to aid solution. Isopropylamine (1 ml) wasthen added. This working solution was warmed on a hotplate (60° C.)whilst purification was ongoing to keep in solution. Injections (1.5 ml)were made using autosampler. Fractions collected on time basis usingfunnel bed fraction collector between 28 min and 35 min. The combinedfraction solutions were evaporated to dryness using a rotary evaporator(30° C. bath temp) and the residue transferred to a tared 20 ml glassvial using ethanol (ca 12 ml). The ethanol was evaporated under a streamof nitrogen gas (room temp).

4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(1.16 g) isolated using the above process was triturated with diethylether (—3 ml) over ˜4 h at ambient temperature. The solid was isolatedby filtration, washed with diethyl ether and dried in vacuo to give4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazoleas a cream solid (0.96 g).

VT NMR (D₆-DMSO): δH 9.03 (s, 1H), 7.98 (s, 1H), 7.74 (s, 1H), 5.43 (m,1H), 4.13 (m, 1H), 4.04-4.00 (m, 6H), 3.61 (m, 2H), 3.46 (m, 1H), 3.28(s, 3H), 2.35 (s, 3H), 2.16 (s, 3H), 2.08 (m, 2H), 1.92 (m, 2H), 1.82(d, 3H). LCMS (formate): Rt 0.76 min, MH⁺451.

Example 34-(8-methoxy-1-((S)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole

Chiral resolution of4-(8-methoxy-1-(1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazolewas carried out using the following conditions:

Column: Chiralpak AD-H (250×30 mm, 5 micron) [ADH10029-01]Flowrate: 45 ml/minDetection: UV DAD (300 nm (bandwidth 180 nm, reference 550 nm (bandwidth100 nm)).

Mobile Phase A: Heptane Mobile Phase B: Ethanol

Isocratic system—85:15 mobile phase A:BRun time—ca. 35 min

4-(8-methoxy-1-(1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(85 mg) was dissolved in ethanol (ca. 4 ml) with heating and sonication.Injections (400 μl) were then made via plastic syringe. The fractionsbetween 24 min and 26.5 min were collected and the combined fractionsolutions were evaporated to dryness using a rotary evaporator (30° C.bath temp). The residue was transferred to a tared glass vial usingethanol (ca 12 ml). The solvent was then removed by evaporation under astream of nitrogen gas (room temp) to give4-(8-methoxy-1-((S)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(38 mg).

VT NMR (D₆-DMSO): δH 9.00 (s, 1H), 7.95 (s, 1H), 7.72 (s, 1H), 5.41 (m,1H), 4.10 (m, 1H), 4.03-3.97 (m, 6H), 3.59 (m, 2H), 3.45 (m, 1H), 3.26(s, 3H), 2.32 (s, 3H), 2.13 (s, 3H), 2.07 (m, 2H), 1.90 (m, 2H), 1.80(d, 3H). LCMS (formate): Rt 0.73 min, MH⁺451.

Example 4 Preparation of a crystalline form of4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(anhydrous form 1)

4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(for a preparation see Example 2 or reaction scheme 1, 18.87 g) wasdissolved in isopropyl acetate (95 mL) at reflux and then allowed tocool to 20° C. over 1 hr. Cyclohexane (190 mL) was then added over 1 hrand the resultant slurry was aged for 1 hr. The slurry was then filteredand washed with 2:1 cyclohexane:isopropylacetate (30 mL) and thencyclohexane (30 mL) before being pulled dry. The cake was then ovendried overnight at 40° C. under vacuum. An XRPD was recorded on thismaterial (see Example 9).

Example 5 Preparation of a crystalline form of4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(anhydrous form 2)

A pre-mixed solution of isopropyl acetate (211 mL) and cyclohexane (422mL) was added to4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(for a preparation see Example 2 or reaction scheme 1, 42.19 g, 94mmol). The resulting suspension was stirred for 24 hours, filtered, theresulting solid washed (1× cyclohexane:isopropyl acetate (2:1, 180 ml),1× cyclohexane (180 ml), 1×TBME (180 ml)), pulled to dryness and driedin vacuo at 40° C. to constant weight giving an almost white solid (90%yield). An XRPD was recorded on this material (see Example 9).

Example 6 Preparation of a crystalline form of4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(hydrate)

A pre-mixed solution of ethanol (2.000 mL) and water (8.00 mL) was addedto4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(for a preparation see Example 2 or reaction scheme 1, 1.00 g, 2.220mmol) and the resulting suspension stirred overnight. The suspension wasfiltered, washed (2× water, 2×TBME), pulled to dryness under vacuum anddried in vacuo at 40° C. to give a white powder. An XRPD was recorded onthis material (see Example 9).

Example 7 Preparation of a crystalline form of4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole(anhydrous form 3)

4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazolehydrate (for a preparation see Example 6, 860 mg, 1.835 mmol) was heatedat 135° C. at 9 mbar overnight. An XRPD was recorded on this material(see Example 9).

Example 8 Preparation of4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazolehydrochloride (hydrochloride)

In a first reactor a solution of7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-4-(N—((R)-1-methoxypropan-2-yl)tetrahydro-2H-pyran-4-carboxamido)quinoline-3-carboxylicacid (for a preparation see Scheme 1, 23.3 kg) in DCM (113 kg) and DIPEA(3.8 kg) was combined with acetonitrile (61.6 kg) and di-iso-propylamine(12.8 kg) the mixture was cooled to between −10 and −3° C.Diphenylphosphoryl azide (19.0 kg) was added maintaining the temperaturebetween −10 and −3° C. and the reaction stirred at this temperature.Further diphenylphosphoryl azide (2.4 kg) was added maintaining thetemperature between −10 and −3° C. and the reaction stirred at thistemperature to form a first solution.

In a second reactor dimethyl formamide (144 kg) and water (72 kg) wereheated to 90-100° C. The first solution was added maintaining thetemperature between 85-100° C., reactor 1 was rinsed with acetonitrile(7 kg) and the rinse added to the second reactor maintaining thetemperature between 85-100° C. The mixture was stirred at thistemperature and then cooled to 20-30° C. The pH was adjusted to pH=10with 30% wt aqueous sodium hydroxide solution (10.4 kg) maintaining thetemperature between 20-30° C. Dichloromethane (315 kg) and water (351kg) were charged, the layers were separated and the aqueous layerextracted with dichloromethane (315 kg). The combined organic layerswere washed with water (234 kg). Water (234 kg) was charged to theorganic layer, the mixture stirred, sodium chloride (80 kg) added andthe layers separated. The organic layer was concentrated under reducedpressure below 30° C., methanol (201 kg) was charged and the solutionconcentrated under reduced pressure below 50° C. Further methanol (199kg) and 4M hydrochloric acid in methanol (68.5 kg) were chargedmaintaining the temperature 20-30° C. and the reaction was stirred atthis temperature. The mixture was concentrated under reduced pressurebelow 50° C. and acetonitrile (104 kg) charged. The mixture wasconcentrated under reduced pressure below 50° C. and furtheracetonitrile (97 kg) charged. The mixture was concentrated under reducedpressure below 50° C. and further acetonitrile (116 kg) charged. Themixture was evaporated under reduced pressure below 50° C. and furtheracetonitrile (103.0 kg) was charged. The mixture was cooled to between−5-0° C. and stirred at this temperature. The slurry was centrifuged intwo portions, each portion was washed with acetonitrile (9 kg) to givethe title compound (33.40 kg, 99.3% purity) as a wet cake.

Example 9 X-ray Powder Diffraction (XRPD) Studies on Crystalline Formsof4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazoleas a free base and as a hydrochloride salt

The data were acquired on a PANalytical X′Pert Pro powderdiffractometer, model PW3040/60 using an X′Celerator detector. Theacquisition conditions were: radiation: Cu Kα, generator tension: 40 kV,generator current: 45 mA, start angle: 2.0° 2θ, end angle: 40.0° 2θ,step size: 0.0167° 2θ, time per step: 31.75 seconds. The sample wasprepared by mounting a few milligrams of sample on a silicon wafer (zerobackground) plate, resulting in a thin layer of powder.

Peak positions were measured using Highscore software. The margin oferror is approximately ±0.1° 2θ for each of the peak assignments. Peakintensities may vary from sample to sample due to preferred orientation.

Table 1 shows characteristic XRPD peak positions and d-spacings forcrystalline form of the compound4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazoleas a free base and as a hydrochloride salt.

TABLE 1 Free base anhydrous Free base Free base Free base form 1anhydrous form 2 anhydrous form 3 Hydrate hydrochloride 2θ/° d-spacing/Å2θ/° d-spacing/Å 2θ/° d-spacing/Å 2θ/° d-spacing/Å 2θ/° d-spacing/Å 7.911.1 6.5 13.6 8.8 10.0 8.1 10.9 9.4 9.4 8.5 10.4 10.8 8.2 11.2 7.9 10.28.7 12.8 6.9 10.7 8.3 13.0 6.8 11.7 7.5 12.0 7.4 13.5 6.6 12.1 7.3 14.06.3 16.1 5.5 14.8 6.0 14.4 6.1 12.7 7.0 15.3 5.8 16.5 5.4 16.6 5.3 14.95.9 13.9 6.4 17.7 5.0 18.2 4.9 17.5 5.1 17.4 5.1 15.9 5.6 19.3 4.6 20.64.3 18.2 4.9 18.9 4.7 16.7 5.3 20.8 4.3 21.5 4.1 18.8 4.7 19.9 4.5 18.84.7 21.6 4.1 23.0 3.9 19.7 4.5 20.4 4.4 21.0 4.2 22.6 3.9 20.5 4.3 24.03.7 22.7 3.9 25.3 3.5 23.3 3.8 25.7 3.5 24.3 3.7 27.2 3.3 24.1 3.7 26.23.4 29.1 3.1 26.6 3.4 32.7 2.7 29.8 3.0

Biological Test Methods

The compounds of formula (I) may be tested in one or more of thefollowing assays.

Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay

The binding of the compounds of formula (I) to Bromodomains BRD2, BRD3and BRD4 was assessed using a time resolved fluorescent resonance energytransfer binding assay, that measures the binding of an acetylatedhistone peptide to the bromodomain protein.

The bromodomain protein, histone peptide and a variable concentration oftest compound are incubated together to reach thermodynamic equilibrium.The assay is configured such that in the absence of test compound thebromodomain and peptide are significantly bound (˜30%) and in thepresence of a sufficient concentration of a potent inhibitor thisinteraction is disrupted leading to a measurable drop in fluorescentresonance energy transfer.

Histone Peptide: H-Ser-Gly-Arg-Gly-Lys(Ac)-Gly-Gly-Lys(Ac)-Gly-Leu-Gly-Lys(Ac)-Gly-Gly-Ala-Lys(Ac)-Arn-His-Gly-Ser-Gly-Ser-Lys(Biotin)-OH. 3TFA

The protected peptide was assembled on a solid-phase synthesiser usingpreloaded Wang resin and utilising standard Fmoc synthesis protocols.The C-terminal lysine was protected by a hyper acid-labile groupallowing for its selective removal at the end of the assembly andattachment of the biotin. The crude peptide was obtained after cleavagefrom the resin with a mixture of trifluoroacetic acid (TFA),triisopropylsilane and water (95:2.5:2.5) for 3 h at room temperatureand was then purified using a C18 reverse-phase column utilising a 0.1%TFA-buffered water/acetonitrile gradient. The resulting fractions wereanalysed and fractions which were >95% pure by analytical HPLC andgiving the correct mw (by MALDiTOF mass spectroscopy) were pooled andfreeze dried. The final material was analysed by HPLC to confirm purity.

Protein Production:

Recombinant Human Bromodomains (BRD2 (1-473), BRD3 (1-435) and BRD4(1-477)) were expressed in E. coli cells (in pET15b vector) with asix-His tag at the N-terminal. The His-tagged Bromodomain was extractedfrom E. coli cells using sonication and purified using a nickelsepharose 6FF column, the proteins were washed and then eluted with 50mM Tris-Hcl pH8.0. 300 mM NaCl, 1 mM 6-mercaptoethanol and 20 mMImidazole. Further purification was performed by affinity chromatographyon a H isTRAP HP column, eluting with a linear 0-500 mM sodium chloridegradient, over 20 column volumes. Final purification was completed bySuperdex 200 prep grade size exclusion column. Purified protein wasstored at −80 C in 20 mM HEPES pH 7.5 and 100 mM NaCl. Protein identitywas confirmed by peptide mass fingerprinting and predicted molecularweight confirmed by mass spectrometry.

Protocol for Bromodomain BRD 2, 3 and 4 Assays:

All assay components were dissolved in buffer composition of 50 mM HEPESpH7.4, 50 mM NaCl and 0.5 mM CHAPS. The final concentration ofbromodomain proteins were 100 nM and the histone peptide was 300 nM,these components are premixed and allowed to equilibrate for 1 hour inthe dark. 8 μl of this reaction mixture was added to all wellscontaining 50 nl of various concentrations of test compound or DMSOvehicle (0.5% final) in Greiner 384 well black low volume microtitreplates and incubated in dark for 60 mins at room temperature. 2 μl ofdetection mixture containing anti-6his XL665 labeled antibody andstreptavidin labeled with europium cryptate was added to all wells and afurther dark incubation of at least 30 mins was performed. Plates werethen read on the Envision platereader, (λex=317 nm, donor λEM=615 nm;acceptor λEM=665 nm; Dichroic LANCE dual). Time resolved fluorescentintensity measurements were made at both emission wavelengths and theratio of acceptor/donor was calculated and used for data analysis. Alldata was normalized to the mean of 16 high and 16 low control wells oneach plate. A four parameter curve fit of the following form was thenapplied:

y=a+((b−a)/(1+(10̂x/10̂c)̂i d)

Where ‘a’ is the minimum, ‘b’ is the Hill slope, ‘c’ is the pI050 and‘d’ is the maximum.

Examples 1-3 were tested in all of the BRD2, BRD3 and BRD4 assaysdescribed above and were found to have a pIC₅₀ in the range 6.5-7.5 ineach assay.

Measurement of LPS induced IL-6 Secretion from Whole Blood

Activation of monocytic cells by agonists of toll-like receptors such asbacterial lipopolysaccharide (LPS) results in production of keyinflammatory mediators including IL-6. Such pathways are widelyconsidered to be central to the pathophysiology of a range ofauto-immune and inflammatory disorders.

Compounds to be tested are diluted to give a range of appropriateconcentrations of which 1 μl of the diluted stocks is added to a 96 wellplate. Following addition of whole blood (130 ul) the plates areincubated at 37 degrees (5% CO₂) for 30 min before the addition of 10 μlof 2.8 ug/ml LPS, diluted in complete RPMI 1640 (final concentration=200ng/ml), to give a total volume of 140 μl per well. After furtherincubation for 24 hours at 37 degrees, 140 μl of PBS are added to eachwell. The plates are sealed, shaken for 10 minutes and then centrifuged(2500 rpm×10 min). 100 μl of the supernatant are removed and IL-6 levelsassayed by immunoassay (typically by MesoScale Discovery technology)either immediately or following storage at −20 degrees. Concentrationresponse curves for each compound was generated from the data and an10₅₀ value was calculated

Examples 1 and 2 were tested in this assay and were found to have a 00₅₀in the range 6.0-7.0.

These data demonstrate that bromodomain inhibitors tested in the abovewhole blood assay inhibited the production of key inflammatory mediatorIL-6.

In Vivo Mouse Model to Demonstrate Modulation of Pro-InflammatoryResponse

Male CD1 mice (25-30 g, n=4 per group) received a single i.v. bolusinjection of LPS (100 pg/kg) via the tail vein 1 h after pre-treatmentwith a single oral administration of either vehicle (1% (w/v)methylcellulose in sterile water) or the test compound (3, 10 and 30mg/kg). Serial blood samples were obtained from the tail vein by directvenepuncture at various time points up to 5 h post-LPS administrationfor analysis of IL-6 and the test compound concentrations by Meso ScaleDiscovery (MSD) analysis and LC-MS/MS respectively.

In vehicle control mice, i.v. LPS induced a time-dependent increase inserum IL-6 concentrations, while mice pre-treated orally with thecompound of Example 2 (3, 10 and 30 mg/kg) had reduced maximum (C_(max))IL-6 levels by 46%, 79%, 73% respectively, and reduced total exposures(AUC) of IL-6 by 35%, 70%, 63% respectively. This reduction in IL-6 iscomparable to the maximal reduction observed with the positive controldexamethasone, i.e.: 73% and 70% reduction in IL-6 C_(max) and AUC,respectively.

These data demonstrate that the compound of Example 2 tested in theabove assay reduced LPS-induced systemic IL-6 levels in the mousefollowing a single oral administration and consequently may have utilityas an anti-inflammatory agent.

Oncology Cell Growth Assay

Human cell lines (n=80 comprising cell lines described in Table 2 below)were cultured in RPMI-1640 containing 10% fetal bovine serum, 1000viable cells per well were plated in 384-well black flat bottompolystyrene plates (Greiner #781086) in 48 μl of culture media. Allplates were placed at 5% CO₂, 37° C. overnight. The following day oneplate was harvested with CellTiter-Glo (CTG, Promega #G7573) for a timeequal to 0 (TO) measurement and compound (20 point titration from 14.7uM to 7 μM) was added to the remaining plates. The final concentrationof DMSO in all wells was 0.15%. Cells were incubated for 72 hours or theindicated time and each plate was developed with CellTiter-Glo reagentusing a volume equivalent to the cell culture volume in the wells.Plates were shaken for approximately 2 minutes and chemiluminescentsignal was read on the Analyst GT (Molecular Devices) or EnVision PlateReader (Perkin Elmer).

Results are expressed as a percent of the TO and plotted against thecompound concentration. The TO value was normalized to 100% andrepresents the number of cells at time of compound addition and theconcentration response data were fit with a 4 parameter curve fit usingXLfit software (model 205). The concentration that inhibited cell growthby 50% (gIC₅₀) is the midpoint of the ‘growth window’ (between the TOand DMSO control). The Ymin−T0 value is determined by subtracting the T0value (100%) from the Ymin value (%) determined from the fit of theconcentration response curve. Values from the wells with no cells weresubtracted from all samples for background correction.

The compound of Example 2 was tested in accordance with the aboveprocedure and found to have the gIC₅₀ as shown in Table 2.

TABLE 2 n (number Cell line type of lines) gIC₅₀ Multiple myeloma 16 1.1to >29326 nM (median 414 nM), with 15 out of 16 cell lines in the range1.1 to 2000 nM Small cell lung cancer 38 76 to >29326 nM (median 538nM), (SCLC) with 35 of the 38 cell lines in the range 76 to 2500 nMCervical cancer 4 266-847 nM NUT-midline carcinoma 2 59-66 nMNeuroblastoma 12 25-435 nM (median 193 nM) Esophageal 8 208-1544 nM(median 759 nM)The compound of Example 2 was also tested in accordance with ananalogous procedure using 96 well plates along with appropriatemodifications to volumes and concentrations that would be apparent tothose skilled in the art. The compound of Example 2 was tested and foundto have the gIC₅₀ as shown in Table 3.

TABLE 3 n (number of Cell line type lines) gIC₅₀ Acute monocyticleukemia 1 123 nM Acute promyelocytic 1 141 nM leukemia Cutaneous T cell1 162 nM lymphoma Burkitt's lymphoma 3 181-807 nM Chronic myeloidleukemia 1 776 nM Breast cancer (ductal) 1 537 nM Ovarian carcinoma 1707 nM

These data demonstrate that the compound of Example 2 tested in theabove assay inhibited cell growth in a panel of oncology cell lines andmay therefore have utility in the treatment of one or more cancers.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

What is claimed is:
 1. A compound of formula (I) which is4-(8-methoxy-1-(1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole

or a salt thereof.
 2. A compound of formula (IA) which is4-(8-methoxy-1-((R)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole

or a salt thereof.
 3. A compound of formula (IB) which is4-(8-methoxy-1-((S)-1-methoxypropan-2-yl)-2-(tetrahydro-2H-pyran-4-yl)-1H-imidazo[4,5-c]quinolin-7-yl)-3,5-dimethylisoxazole

or a salt thereof.
 4. A compound according to claim 2 or apharmaceutically acceptable salt thereof.
 5. A pharmaceuticalcomposition which comprises a compound or a pharmaceutically acceptablesalt thereof as defined in claim 4 and one or more pharmaceuticallyacceptable carriers, diluents or excipients.
 6. A combinationpharmaceutical product comprising a compound or a pharmaceuticallyacceptable salt thereof as defined in claim 4 together with one or moreother therapeutically active agents. 7-11. (canceled)
 12. A method oftreating diseases or conditions for which a bromodomain inhibitor isindicated in a subject in need thereof which comprises administering atherapeutically effective amount of a compound or a pharmaceuticallyacceptable salt thereof as defined in claim
 4. 13. A method of treatmentaccording to claim 12, wherein the disease or condition is a chronicautoimmune and/or inflammatory condition.
 14. A method of treatmentaccording to claim 12, wherein the disease or condition is cancer.
 15. Amethod of treatment according to claim 12 in which the subject is ahuman.